6.2.1 Outside the Incubator

Outside the incubator, gametes and embryos are exposed to light, variable and nonphysiological temperatures and pH, airflow, and varying air quality. Exposure begins at oocyte retrieval and ends with embryo transfer and/or cryopreservation.

IVF requires varying degrees of micromanipulation, e.g., intracytoplasmic sperm injection (ICSI), hatching, and embryo biopsy. As technique complexity has increased, gametes and embryos are exposed more to the environment outside the incubator.

A single study mapped the time required by laboratory staff to attend to each of three IVF protocols: traditional IVF (350 min), contemporary IVF (520 min), and IVF that incorporates preimplantation genetic testing (780 min).⁴ To place this study into context with this chapter, these estimated attention or personnel workload times highlight the additional time oocytes/embryos must be handled outside the incubator according to procedure complexity. Additional care is required to protect gametes/embryos during these extended work times.

6.2.2 Inside the Incubator

Incubators are classified as big- and small-box, benchtop small-chamber, and time-lapse (benchtop small-chamber incubator with a camera system) types. There are positive and negative features of each⁵ (reviewed in Chapter 2). Oocytes/embryos spend the bulk of their laboratory existence in one of these devices, and some time out of incubation.⁴

6.3.1 Bicarbonate-Buffered Media

The generation of hydrogen ions and their stabilization with bicarbonate ions in culture media has been described in detail¹⁵ and is summarized in Figure 6.1. Carbon dioxide is elevated in the culture environment, either in the incubator chamber or within a small enclosure, such as an isolette, funnel, bell jar–type lid, or microscope chamber. Carbon dioxide from the environment combines with water in the medium to produce carbonic acid, a weak acid that does not dissociate to completion. The products of this dissociation – hydrogen ions and bicarbonate ions – reassociate in the reverse direction to generate carbonic acid and these reactions rapidly reach an equilibrium, one that is defined by an acid dissociation constant, Ka. Sodium bicarbonate is included as a medium component, typically in the range of 20–25 mM, and acts to stabilize excessive ionization of carbonic acid. Stable hydrogen ion concentrations within the medium are then attained by adjusting the atmospheric CO₂ level that controls the amount of carbonic acid produced and, ultimately, hydrogen ion levels. Addition of more CO₂ into the atmosphere produces more carbonic acid, thus reducing pH, whereas lowering CO₂ levels has the reverse effect.

Figure 6.1 Stabilization of hydrogen ions with bicarbonate anions in culture media.

One principle the embryologist must keep in mind is that the amount of hydrogen ion generated depends upon the number of CO₂ molecules present in the atmosphere, and this number changes as a function of altitude. In other words, the number of CO₂ molecules present in an atmosphere of 5% CO₂ at sea level is not the same as it is at 1000 meters above sea level; therefore, the percent of CO₂ utilized is a relative term and adjustments must be made according to the altitude where the laboratory is situated. The easiest way to estimate the requirement is to use the Henderson–Hasselbalch equation – this is shown in Figure 6.2. Atmospheric pressure, expressed in mm of Hg, is used to calculate the partial pressure of CO₂.

Figure 6.2 The Henderson–Hasselbalch equation. For color version of this figure.

A second important consideration concerns if and how the medium is supplemented with protein or any additional additives in the laboratory. Most albumin and other protein supplements are prepared and distributed in normal saline (0.9% sodium chloride). When an albumin solution is added, volume to volume, to produce a given working protein concentration, medium components are reduced in concentration by the same amount. This includes sodium bicarbonate and will reduce pH. For example, addition of 0.5 mL of a 100 mg/mL solution of human albumin to 9.5 mL of culture medium to yield a working solution of 5 mg/mL of albumin will yield a culture medium with a 5% reduction in all medium components, an effect that may be measurable in terms of buffer capacity. It is therefore crucial in quality control to measure the complete, supplemented culture medium for pH determinations in order to obtain an accurate reading.

6.3.2 Zwitterionic-Buffered Media

Working with bicarbonate-buffered media outside the incubator is labor-intensive as the surroundings must be controlled for appropriate CO₂ levels in order to provide stable and correct pH levels. A simpler alternative is to use media containing zwitterionic compounds, thus negating the need for additional CO₂ outside the incubator. Zwitterionic compounds are hybrid molecules containing both acidic and basic groups that can dissociate as either an acid or a base, depending upon the pH of the solution. In this manner, changes in pH are moderated, adding buffer capacity to the solution. One of the best known and most effective zwitterionic buffers employed in biological systems is N-2-hydroxy-ethylpiperizine-N′-2-ethanesulfonic acid, abbreviated HEPES. This, along with 11 other zwitterionic compounds, was developed by Good et al. (1966) as an alternative to inappropriate buffers, such as phosphate buffer, that were inefficient and produced undesired reactivity or toxicity.¹⁶ HEPES is a substituted taurine and provides excellent buffer capacity in concentrations of 20–25 mM. A second zwitterionic buffer, 3-(N-morpholine) propane sulfonic acid, abbreviated MOPS, has also been used quite effectively in handling media for oocytes and embryos, also in the range of 20–25 mM.

One important principle in selecting an appropriate zwitterionic buffer is to recall that the pKa of a buffer is temperature sensitive and that selecting a buffer where the pKa is closest to the desired working pH of the solution yields the maximum buffering capacity. The pH buffering range of HEPES is 6.8 to 8.2 with a pKa at 25°C of 7.48 and at 37°C of 7.31. MOPS has a buffering range of 6.5–7.9 with a pKa at 25°C of 7.2 and at 37°C of 7.02. When working at near room temperature, MOPS would offer better buffer capacity, but the choice shifts to HEPES at 37°C, given their respective pKas at those temperatures. One approach to negating the need to make individual buffer selections as a function of working temperature was developed by Swain and Pool (2009) who formulated handling media containing both MOPS and HEPES at concentrations of 10 mM each.⁷ This concept of temperature independence through the formulation of a multi-protic buffer has been employed by several commercial vendors in both oocyte and gamete handling media along with vitrification solutions.

6.3.3 Equilibration of Media

Ensuring that bicarbonate-buffered media have been charged with a sufficient amount of CO₂ to produce the desired target pH for embryo handling and growth is a standard component of effective laboratory quality control. The time required for this process is dependent upon several factors including the culture volume and configuration (surface area), volume of oil overlay, oil type and permeability to CO₂, and atmospheric concentration of CO₂. Incubator chamber volume, gas recovery time, and frequency of chamber openings are also important in terms of knowing that the equilibrated levels remain constant. A recurring mistake made in many laboratories is failure to measure accurately or to use consistent oil volumes for culture. When this occurs, equilibration times may change for each dish prepared. Once the appropriate equilibration time has been validated for the culture system employed, a standard time of day can be set aside for this procedure, thus assuring repeatable buffer capacity.

6.4.1 Outside the Incubator

Optimal handling and culture temperatures are still debated – benches, work tables, tube warmers, microscope surfaces, and inverted microscope stages are modified to maintain a desired temperature: Remember, however, that the temperature will not be static but rather cyclical around a set point. The cytoskeleton of oocytes is sensitive to cooling and wide and frequent fluctuations in temperature.¹⁷ As routinely used, the target temperature for heated surfaces should be 37.0°C. Diligent monitoring is essential to identify hot spots, cold spots, and changes over time. Also, surface temperature may not be the same as inside a tube, or in a microdrop at the bottom of a dish. But the stability of the oocyte spindle apparatus with dissolution (chemical or temperature) and reformation is robust, and, with healthy oocytes, ICSI can be performed successfully at room temperature¹⁸ Exposing oocytes and embryos to higher temperatures can be more damaging than cooling, e.g., compromising genetic expression.

6.4.2 Inside the Incubator

Incubation temperature for human oocytes and embryos has historically been targeted to 37.0°C, a presumed central measurement of human body temperature. In vivo temperature gradients across the ovary to the uterus have been documented in various mammals, suggesting that metabolic and other cellular requirements may vary according to site.¹⁹

Optimal incubation temperatures are species dependent, but there are only a few well-designed studies regarding incubation temperature and human IVF. Hong et al (2014) evaluated incubation at 36°C and 37°C.²⁰ Multiple incubators were used, and incubator stability was 36 ± 0.07°C and 37 ± 0.04°C. Fertilization and embryonic aneuploidy rates were not significantly different, but cleavage-stage cell numbers, blastocyst conversion, and “usable” blastocyst numbers were greater with incubation at 37°C compared to 36°C. There were no differences in per embryo implantation rates. Another study evaluated incubation temperatures of 36.5°C and 37°C and found no significant effects of temperature on outcomes, e.g., implantation rate or clinical pregnancy, in a large study population.²¹ But while embryo cleavage rates were higher at 36.5°C, descriptive embryo quality was lower overall on day 3, and blastocyst conversion rate (formation and numbers available for cryopreservation) and descriptive blastocyst quality were significantly lower. Both studies concluded that despite the relatively narrow differences in culture temperatures, the target temperature of 37°C was overall the best choice for the human embryo.^{20, 21}

Incubators maintain a somewhat steady-state temperature, but the actual temperature, like gas phase transitions, will be cyclical. In addition, metabolic fluctuations could occur by extended or repeated periods of cooling (outside the incubator) and then rewarming (reintroduction to incubation). Dishes and tubes of various sizes and design, use of oil overlay, and movement to warm surfaces or tube holders can limit temperature fluctuations outside of incubation. Depending on incubator design, the time required for return to target temperature varies.⁵

Digital readouts on incubators can deviate from the “true” or actual temperature. While oocytes and embryos can tolerate (some) cooling, higher temperatures, e.g., over 38.0°C, inside or outside of the incubator, should be avoided. Choi et al. (2015) exposed one-cell mouse embryos to elevated temperatures (37, 39, 40, and 41°C) for short (8 hours) and long-term (96 hours) intervals.²² Severe, short-term heat stress compromised early cleavage, and trophectoderm cell number and quality was diminished with long-term heat stress. Gene expression was altered, as were post-transfer fetal metrics. Youssef et al (2016) examined the culture temperature for mouse embryo culture.²³ Variable blastocyst development and hatching blastocyst rates were observed at 36, 37, 37.5, 38, and 39°C. Blastocyst hatching was highest at 37.5°C, but combined development and hatching rate was higher at 37°C.